This invention relates to reaction vessels useful for heat exchanging chemical processes, such as polymerase chain reaction (PCR).
Conventional PCR instrumentation typically consists of a block of aluminum having as many as ninety-six conical reaction tubes. The aluminum block is heated and cooled either by a Peltier heating/cooling apparatus, or by a closed-loop liquid heating/cooling system, flowing through channels machined into the aluminum block.
A fundamental element of the PCR process is thermal cycling, during which the temperature of the aluminum block is cycled between 60.degree. C. and 95.degree. C. as often as fifty times. Because of the large thermal mass of the aluminum block, heating and cooling rates are limited to about 1.degree. C./sec, so that a fifty cycle PCR process may require two or more hours to complete.
The highest heating rate in commercial instruments is on the order of 5.degree. C./second, and cooling rates are significantly less. With these relatively slow heating and cooling rates, it has been observed that some processes, such as PCR, are inefficient. For example, reactions may occur at the intermediate temperatures, creating unwanted and interfering DNA products, such as "primer-dimers" or anomalous amplicons, as well as consuming reagents necessary for the intended PCR reaction. Other processes, such as ligand binding, organic or enzymatic, when performed in non-uniform temperature environments, similarly suffer from side reactions and products that are potentially deleterious to the analytical method.
Another fundamental element of the PCR process is detection of the amplified DNA molecules. PCR-produced DNA has usually been detected in a second, complex, heterogeneous chemical hybridization step. New homogeneous DNA detection techniques, particularly ethidium bromide and polymerase chemistries based upon optical fluorescence, are providing PCR with new, highly specific, quantitative capabilities. One major advantage of this new chemistry is that accurate, quantitative data can be attained by monitoring optical fluorescence after each thermal cycle. In terms of practical implementation, the most important aspect is that complex, heterogeneous reactions are not required since the detection chemistry is performed in the PCR reaction chamber without further sample handling.
Preferred detection techniques for the analysis of DNA, RNA, and other biologicals are optical, typically fluorescence or chemiluminescence. Optimum optical sensitivity of fluorescence activity in fluid solutions can be attained by maximizing the optical sampling path-length of both the light beams exciting the fluorescence molecules and the emitted light that will be detected to generate the optical signal.
Instrumentation suitable for these newer processes, requiring "real-time" optical analysis after each thermal cycle, has only recently become available. Examples of these instruments include the Perkin Elmer 7700 (ATC) instrument and the Perkin Elmer 9600 instrument. Both instruments employ a 96-well aluminum block format, however, so that their heating and cooling rates are relatively slow. Optical fluorescence detection in the PE 7700 is accomplished by guiding an optical fiber to each of the ninety-six reaction sites. A central high power laser sequentially excites each reaction tube and captures the fluorescence signal through the optical fiber. Since all the reaction sites are sequentially excited by a single laser and fluorescence is detected by a single spectrometer and photomultiplier tube, complex beam-guiding and optical multiplexing are required.
An instrument from Idaho Technologies monitors each reaction tube in sequence as capillary sample carriers are rotated past heating and cooling sites and optical interrogation sites. This instrument is much simpler that the ATC, however, it is not easily configured for commercial, high throughput PCR diagnostic applications.
A third real-time PCR analysis system is the MATCI device developed by Dr. Allen Northrup et al., as disclosed in U.S. Pat. No. 5,589,136, incorporated herein by reference. This device uses a modular approach to PCR thermal cycling and optical analysis. Each reaction is performed in its own thermal cycling sleeve and each sleeve has its own associated optical excitation source and fluorescence detector. Using a new generation of blue LED's, simple optics and solid-state detectors, real-time data can be obtained from a compact, low-power module. Not only are the optics simple and efficient, but the low thermal mass of the thermal cycling sleeve allows the MATCI device to realize extremely fast thermal heating and cooling rates, up to 30.degree. C./sec heating and 5.degree. C./sec cooling.
For some diagnostic and environmental applications of PCR and other chemical detection methodologies, the volume of the tested unknown sample can be important. For example, in the detection of viruses in blood or urine, if a detection sensitivity of 10 virions/mL is necessary, then, a minimum fluid volume of at least 0.1 mL is required. (Statistically, 0.1 mL will only reliably detect about 30-40 virions/mL.) Therefore, the chemical analysis system must be designed to handle a wide range of fluid volumes, from nanoliters to milliliters.
For these reasons, optimization of the PCR process and similar biochemical reaction processes requires that the desired optimal reaction temperatures be reached as quickly as possible, spending minimal time at intermediate temperatures. Therefore the reaction vessels containing the reactants must be designed to optimize heating and cooling rates, to permit real time optical interrogation, and to accept various sample volumes.